The present invention relates to a method for measuring the concentration or the activity of urinary trypsin inhibitor (UTI) contained in body fluids. The presence of UTI was initially recognized in urine, but its presence has been confirmed in body fluids other than urine by subsequent studies. Therefore, the present invention can be applied to body fluids other than urine, for example, blood serum, blood plasma, cerebrospinal fluid, amniotic fluid, and other appropriate body fluids.
UTI was discovered by Bauer and Reich in 1909 as a trypsin inhibitor which is present in urine. Since this, it has been reported that the amount of UTI present in urine significantly increases in patients with bacterial infections, malignant tumors (for example, gastric cancer, breast cancer, lung cancer), renal diseases or myocardial infarction, or patients having undergone surgical operations, pregnancy, or the like. Particularly in the field of pediatrics, its usefulness as an early indicator of bacterial infections has been noted (Japanese Journal of Inflammation 14: 53-57, 1994).
Conventionally, the activity or the concentration of UTI has been measured by enzymatic methods by measuring the inhibition of trypsin activity, or by various immunological methods based on the reaction between UTI and anti-UTI antibodies. Examples of immunological measuring methods include single radial immunodiffusion (SRID), radioimmunoassay (RIA), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), latex agglutination immunoassay (LAIA), and the like.
In the above-mentioned enzymatic measuring methods, rapid measurement is enabled by the use of an automatic analyzer. However, because the inhibition of trypsin activity is measured, trypsin inhibitors other than UTI are also measured, so that there are problems in specificity. On the other hand, in the above-mentioned immunological measuring methods, because the measurement is based on antigen-antibody reaction, UTI alone can be measured specifically.
However, there are various problems in conventional immunological measuring methods. First, RIA has a problem in that the measurement can be carried out only in special facilities because of the use of radioactive materials. Furthermore, in SRID, EIA and ELISA, the operation of the measurement is complicated and also it requires a lot of time. On the other hand, in LAIA, measurement can be carried out easily within a short time, but if a normal automatic analyzer is used, the measured values may vary due to non-specific agglutination of latex, or the tubes and the like of the automatic analyzer may become clogged with dried reagents.
Thus, the usefulness of UTI as an indicator of various diseases has been recognized, but there are some problems in the measuring methods and so it has not been utilized sufficiently. Therefore, it is an object of the present invention to provide a method by which UTI can be measured rapidly and easily with high precision.
In order to achieve this object, the present invention provides a method for measuring the concentration of UTI in a sample, said method comprising preparing antibodies against UTI that are not adhered to an insoluble support (free anti-UTI antibodies), adding the antibodies to said sample, and measuring the degree of the resulting agglutination. Examples of the insoluble support include latex particles, gold colloid particles, and the like.
Thus, because the method of the present invention utilizes antigen-antibody reaction with excellent specificity, it is excellent in precision and reproducibility. Furthermore, because the degree of agglutination generated by the antigen-antibody reaction is measured, the measurement can be carried out easily without need of special operations such as immobilization of antibodies, or use of special equipment or apparatuses. Furthermore, compared with LAIA, it has an advantage of causing less contamination in an automatic analyzer.
The point of the present invention is that, with respect to UTI, it was found that agglutination reaction can be measured even if free anti-UTI antibodies that are not adhered to an insoluble support such as latex particles etc. are used. By not adhering antibodies to latex particles etc., the present invention has solved the problems of LAIA, and thus UTI can be measured speedily and easily with high precision.
In the method of the present invention, it is preferable to add antibodies into a sample in the presence of 2 to 10 weight % of polyethylene glycol in the reaction solution. The polyethylene glycol is used as an agglutination accelerator, which enables the measurement to be carried out more rapidly and accurately. By xe2x80x9cthe reaction solutionxe2x80x9d is understood an antigen-antibody reaction solution, namely, a solution containing a sample and free anti-UTI antibodies.
Preferably, the average molecular weight of the polyethylene glycol is in the range of 2,000 to 20,000.
In a particularly preferred embodiment, the average molecular weight and the concentration of the polyethylene glycol in the reaction solution are preferably in the range of 6,000 to 20,000 with respect to average molecular weight and 4 to 6 weight % with respect to concentration.
Any means of determining the degree of agglutination may be used. However, in the method of the present invention, the degree of the agglutination is preferably measured by an optical method. Although it is possible to measure the degree of the agglutination by visual observation, the measurement can be conducted more rapidly and accurately by optical measuring. In the optical measuring, for example, as mentioned below, the change in absorbance depending on the degree of agglutination is preferably measured. An alternative example of a measuring method other than optical measuring is an electrical resistance measuring method, in which the change in electrical resistance caused by the degree of agglutination is measured.